Research Article |
Corresponding author: Qi Lin Tang ( tangqilin71@163.com ) Academic editor: Ekaterina Badaeva
© 2018 Muhammad Zafar Iqbal, Mingjun Cheng, Yanli Zhao, Xiaodong Wen, Ping Zhang, Lei Zhang, Asif Ali, Tingzhao Rong, Qi Lin Tang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Iqbal MZ, Cheng M, Zhao Y, Wen X, Zhang P, Zhang L, Ali A, Rong T, Tang QL (2018) Mysterious meiotic behavior of autopolyploid and allopolyploid maize. Comparative Cytogenetics 12(2): 247-265. https://doi.org/10.3897/CompCytogen.v12i2.24907
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This study was aimed to investigate the stability of chromosomes during meiosis in autopolyploid and allopolyploid maize, as well as to determine an association of chromosomes between maize (Zea mays ssp. mays Linnaeus, 1753) and Z. perennis (Hitchcock, 1922) Reeves & Mangelsdor, 1942, by producing a series of autopolyploid and allopolyploid maize hybrids. The intra-genomic and inter-genomic meiotic pairings in these polyploids were quantified and compared using dual-color genomic in-situ hybridization. The results demonstrated higher level of chromosome stability in allopolyploid maize during meiosis as compared to autopolyploid maize. In addition, the meiotic behavior of Z. perennis was relatively more stable as compared to the allopolyploid maize. Moreover, ten chromosomes of "A” subgenome in maize were homologous to twenty chromosomes of Z. perennis genome with a higher pairing frequency and little evolutionary differentiation. At the same time, little evolutionary differentiation has been shown by chromosomes of "A” subgenome in maize, while chromosomes of "B” subgenome, had a lower pairing frequency and higher evolutionary differentiation. Furthermore, 5IM + 5IIPP + 5IIIMPP and 5IIMM + 5IIPP + 5IVMMPP were observed in allotriploids and allotetraploids respectively, whereas homoeologous chromosomes were found between the "A” and "B” genome of maize and Z. perennis.
Maize, polyploidy, meiosis, GISH, chromosome stability, genome evolution
Zea Linnaeus, 1753, belongs to the tribe Maydeae Candolle, 1882, and consists of two sections: section Luxuriante and section Zea (
From previous research, it is evident that crosses could be made between maize and Z. perennis; as a consequence the genomic relationship was assessed by meiotic pairing analysis of hybrids between both species (
GISH Genomic in situ Hybridization
RCC Relative chaotic coefficient
PMCs Pollen mother cells
Plant materials are shown in Table
The roots collected from parents and interspecific hybrids were immediately fixed in a saturated solution of α-bromonaphthalene for three hours, subsequently, transferred in Carnoy’s solution I (3:1 ethanol: glacial acetic acid, v/v) for 24 hours and, finally submerged in 70% ethanol solution after which these were preserved at 4 °C. Pre-mature anthers of hybrids and parents were collected and treated with Carnoy’s solution for a minimum of 12 hours and then preserved in 70% ethanol solution at 4 °C.
The preserved root tips and anthers were cleaned with distilled water to remove the effects of ethanol and then treated with an enzymatic solution comprising 6% cellulase (R-10, Yakult, Japan) and 1% pectinase (Y-23, Yakult, Japan) for 2.5–5.0 hours at 37 °C. Root tips and anthers were again thoroughly cleaned with distilled water in order to wash enzyme solution and finally, squashed onto glass slides in a drop of Carnoy’s solution I and dried with ethanol flame. The preparations showing well-spread and clean mitotic and meiotic chromosomes were selected by phase-contrast light microscopy (Olympus BX-41, Japan) and stored at -20 °C for in situ hybridization. Total genomic DNA from young leaves of maize and Z. perennis was extracted according to modified 2 × CTAB methods (
The genomic DNA of maize and Z. perennis were labeled with DIG-Nick Translation and BIOTIN-Nick Translation Mix (Roche, Swiss), respectively according to manufacturer’s protocol. The selected slides were preheated in an air blowing oven at 60 °C for one hour and then transferred into 0.1ug/ml RNase (Solarbio, China) in 2 × SSC solutions in a thermostat water bath at 37 °C for one hour. Afterwards, slides were washed twice in 2 × SSC for 5 minutes each at room temperature, followed by chromosome denaturation in 70 percent deionized formamide (FAD) solution at 70 °C for 2.5 minutes, then immediately dehydrated in an ice-cold 70 percent, 95 percent and 100 ethyl alcohol series and finally air dried at room temperature. The hybridization mixture comprised 150 µl 50% FAD, 60 µl 10% dextran sulfate (DS), 30 µl 2 × SSC, 15 µl 0.5% sodium dodecyl sulfate (SDS), 30 μg salmon sperm DNA (SSDNA) and 18 µl labeled probes for six slides. Hybridization mixture was denatured in a thermostat at 85 °C for 10 minutes, followed by quick cooling in ice for 10 minutes. A total 46 μl hybridization mixture was loaded on each slide and hybridization was accomplished in an incubator at 37 °C for 20–24 hours. After hybridization slides were immersed in 20% FAD, 2 × SSC, 0.1 × SSC, respectively for 15 minutes each, at 42 °C. After that, the slides were washed in 0.1% Triton X-100 once and in 1 × PBS thrice for 5 minutes each and then air dried, at room temperature. All further steps were performed in dark, 50 µl antibody diluent, which contained anti-digoxigenin-fluorescein (0.6 µg/µl in 1 × PBS, Roche) and streptavidin-Cy-3 fluorescein (0.6% in 1 × PBS, Sigma) was applied onto air dried slides and immunodetection was done at 37 °C for one hour in an incubator. Consequently, slides were washed in 1 x PBS thrice for 5 minutes each at room temperature and air dried finally. The chromosome counterstaining was performed by 4, 6-diamidino-2-phenylindole (DAPI) solution containing 86% 1 × PBS and 14% DAPI 10ug/ml (Solarbio), and slides were observed with fluorescence microscope (Olympus BX-61, Japan) coupled with pre-fixed filter sets named as U-MNAU2 (excitation 360–370nm; emission 420–460nm and dichroic 400nm), MWIBA3 (excitation 460–495nm; emission 510–550nm and dichroic 505nm) and U-MWIG3 (excitation 530–550nm; emission 575nm IF and dichroic 570nm). The images were captured with Media Cybernetics CCD 700 (Charge Coupled Device) and Image Pro Plus 6.0 (Media Cybernetics, Inc.). Captured images were processed by Adobe Photoshop 5.1.
Three crosses were made between diploid maize, tetraploid maize and Z. perennis (9475) to produce polyploid hybrids, and those synthetics are shown in Fig.
The schematic sketch of "U” triangle presents the production strategy of polyploid hybrids from one-way crosses of diploid and tetraploid parent (wf9, Twf9 and 9475) . The maize and Z. perennis cytoplasm are represented by light green and light pink circles, respectively. The dense green and dense pink strips represent maize and Z. perennis chromosomes, respectively and central red marks represents centromere of both types of chromosome
Composition of chromosomes in hybrids revealed by carbol fuchsin staining and GISH. a, b, c, d represents chromosome counts of wf9, MM30, Twf9, and Z. perennis. e + f and g + h represent chromosomal composition of MP30 and MP40, respectively. Yellow and pink signals represent maize and Z. perennis genome, respectively. All bars = 10 µm. The blue terminal ends of maize chromosomes represent maize knobs (intensely stained with DAPI).
Diploid maize genome exhibited regular meiosis and the most frequently observed meiotic configuration was 10II (Fig.
Chromosome pairing analysis of parents and hybrids. a, b, c, d, e (e1), f (f1) represent diakinesis of wf9, 9475, MM30, Twf9, MP30 and MP40, respectively. a1, b1, c1, d1, e2, f2 represent meiotic anaphaseIand e3, f3 represent meiotic telophaseI. Black arrow represents univalent, blue arrow represents bivalent, green arrow represents trivalent, yellow arrow represents quadrivalent, red arrow represents quadrivalent in diploid maize. White triangle represents univalent of maize genome. Z. perennis and maize autosyndetic bivalents are shown by blue and purple triangles, respectively. The allosyndetic bivalents, which were composed of one maize and one Z. perennis chromosome are represented by red triangles. The allosyndetic trivalents consisting of one maize and two Z. perennis chromosomes are represented by green triangles. Allosyndetic quadrivalents composed of two maize and two Z. perennis chromosomes are indicated by yellow triangle, while white arrow indicates lagging chromosomes g, h, i, j, k, l, m, n show different pairing types with the models below. Yellow and pink signals represent maize and Z. perennis genomes, respectively. All Bars = 10 µm.
The most prevalent meiotic configuration of MM30 was 1I+4II+7III (29.67%) with the average of 0.71I+3.31II+7.19III+0.28IV, while 10III (11.72%) was also found in some PMCs (Table
Average number of meiotic chromosomes associations in PMCs of triploid hybrids verified by GISH.
Hybrids | 2n | I | II | III | IV | RCC | PMCs | |||||||||
Total | IM | IP | Total | IIMM | IIPP | IIMP | Total | IIIMPP | Others | Total | Total | wf9 | 9475 | |||
MM30 | 30 | 0.71b | 0.71b | – | 3.31b | 3.31a | – | – | 7.19a | – | 0.28a | 4.78a | 4.78b | – | 129 | |
MP30 | 30 | 4.56a | 3.79a | 0.77 | 5.44a | 0.25b | 4.34 | 0.85 | 4.76b | 4.55 | 0.21 | 0.07b | 2.59b | 9.00a | 1.56 | 71 |
The PMCs of MP30 frequently showed lagging chromosomes at meiotic anaphase I (Fig.
For more detailed analysis of chromosomes in MP30, the configuration of IIIMPP was examined. The configuration of IIIMPP was not similar in all cases (Fig.
The most frequent meiotic configuration of MM40 was 10IV (21.67%) with the average of 0.26I+3.61II+0.14III+8.03IV (Table
Average number of chromosomes associations in PMCs of tetraploid hybrids revealed by GISH.
Hybrids | 2n | I | II | III | IV | RCC | PMCs | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Total | IM | IP | Total | IIMM | IIPP | IIMP | Total | Total | IVMMPP | Others | Total | wf9 | 9475 | |||
MM40 | 40 | 0.26b | 0.26b | – | 3.61b | 3.61b | – | – | 0.14a | 8.03a | – | – | 4.83a | 4.83a | – | 121 |
MP40 | 40 | 1.17a | 0.81a | 0.36 | 9.97a | 4.30a | 4.71 | 0.96 | 0.13a | 4.62b | 4.29 | 0.33 | 1.46b | 1.60b | 1.47 | 69 |
The most common meiotic configurations of allotetraploid maize (MP40) were 8II+6IV (15.94%) and 12II+4IV (15.94%), and the lagging chromosomes found at meiotic anaphase I (Fig.
The detailed chromosome observation of MP40 showed that configurations of IVMMPP were not similar. The most frequent configuration of IVMMPP was of "ring type” with an average number (range) of 0.72 (0–6), while another form of "rod type” also found (Fig.
The comparative analysis of MM40 and MP40 revealed that the RCC of maize genome in MM40 was higher than MP40, suggested that a limited homology between maize and Z. perennis genomes enhance meiotic stability in maize allotetraploid. Comparative analysis between Z. perennis and MP40 showed higher number of bivalents and lower RCC in Z. perennis than MP40 and Twf9 that might be due to allopolyploid nature of Z. perennis (
Polyploidy is a state in which more than two sets of chromosomes coexist in one nucleus. It is a widespread phenomenon in plants and is considered to be a major force in plant evolution (
The maize genome has a large number of duplicated genes according to theory of tetraploid origin (
Materials | wf9 | 9475 | MM30 | MP30 | MM40 | MP40 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Average configuration | 0.01I+7.31II +0.01III+1.33IV | 0.18I+10.46II +0.13III+4.62IV | 0.71I +3.31II +7.19III+0.28IV | 4.56I+5.44II +4.73III+0.07IV | 0.26I+3.61II +0.14III+8.03IV | 1.17I+9.97II +0.13III+4.62IV | |||||||||
Frequent configurations | 10II (35.00) | 10II+5IV (34.83) | 1I+4II+7III (29.67) | 10III (11.72) | 5I+5II+5III (16.9) | 10IV (21.67) | 8II+6IV (15.94) | 12II+4IV (15.94) | 10II+5IV (13.04) | ||||||
Frequent valents (Range) | I (%) | Total | 0 (98.77) | 0 (88.56) | 0 (51.9) – | 5 (25.3) | 0 (80.83) | 0 (52.1) | |||||||
IM | 0 (98.77) | (0–1) | 0 (51.9) | (0–3) | 5 (18.3) | (0–8) | 0 (80.83) | (0–2) | 0(55.07) | (0–5) | |||||
IP | – | – | 0 (88.56) – | (0–4)– | – | 0(57.75) | (0–7) | – | – | 0(71.01) | (0–2) | ||||
II (%) | Total | 8 (33.33) | 10 (37.3) | 4 (24.0) | 5 (29.58) | 0 (25.00) | 10 (17.3) | ||||||||
IIMM | 8 (33.33) | (0–10) | 4 (24.0) | (0–9) | 0(74.65) | (0–1) | 0 (25.00) | (0–10) | 5(24.64) | (2–7) | |||||
IIPP | – | – | 10 (37.31) – | 15 | – | 5(26.76) | (1–7) | – | – | 5(27.54) | (1–9) | ||||
IIMP | – | – | – | – | – | 0(52.11) | (0–4) | – | – | 0(59.42) | (0–4) | ||||
III (%) | Total | 0 (98.77) | 0 (90.55) | 8 (24.0) | 5 (33.8) | 0 (86.67) | 0(88.41) | ||||||||
IIIMMM | 0 (98.77) | (0–1) | 8 (24.0) | (2–10) | 0 (95.2) | (0–1) | 0 (86.67) | (0–2) | 0 (100) | ||||||
IIIPPP | – | – | 0 (90.55) | (0–3) – | – | 0(90.14) | (0–1) | – | – | 0(94.20) | (0–1) | ||||
IIIMMP | – | – | – | – | – | 0(91.55) | (0–3) | – | – | 0(97.10) | (0–1) | ||||
IIIMPP | – | – | – | – | – | 5(36.62) | (0–7) | – | – | 0(94.20) | (0–1) | ||||
IV (%) | Total | 1 (34.57) | 5 (40.30) | 0 (77.5) | 0 (92.96) | 9 (24.17) | 5 (26.09) | ||||||||
IVMMMM | 1 (34.57) | (0–5) | 0 (77.5) | (0–3) | 0 (100) | 9 (24.17) | (4–10) | 0(81.16) | (0–3) | ||||||
IVPPPP | – | – | 5 (40.30) – | (1–8) | – | 0(97.18) | (0–1) | – | – | 0(89.86) | (0–1) | ||||
IVMMPP | – | – | – | – | – | 0(95.77) | (0–1) | – | – | 5(24.64) | (1–7) |
Chromosome pairings between maize and Z. perennis was observed in PMCs of two allopolyploids. We found univalents, bivalents and multivalents and allosyndetic valents at different levels during meiosis. The meiotic configuration of MP30 was 5IM+5IIPP+5IIIMPP, while univalents IM, bivalents IIPP and allosyndetic trivalents IIIMPP were common. The meiotic configuration of MP40 was 5IIMM +5IIPP +5IV, while bivalents IIMM, bivalents IIPP and allosyndetic quadrivalents IVMMPP were frequently observed (Table
Detailed examination of allosyndetic trivalents (IIIMPP) revealed that there were not only "frying pan type”, but "rod type” also existed in allotriploid with a maximum number of five (Table
Valente types | MP30 | MP40 | |||
IIIfry-pan type | Mean (Range) | 3.23 (0–6) | – | ||
Frequency (%) | 3 (32.39) | – | |||
4 (18.31) | – | ||||
5 (22.54) | – | ||||
IIIrod type | Mean (Range) | 1.18 (0–5) | – | ||
Frequency (%) | 0 (33.80) | – | |||
1 (30.99) | – | ||||
2 (23.94) | – | ||||
IVring type | Mean (Range) | – | 2.78 (0–6) | ||
Frequency (%) | – | 2 (26.76) | |||
– | 3 (26.76) | ||||
– | 1,4,5 (14.08) | ||||
IVrod type | Mean (Range) | – | 0.72 (0–6) | ||
Frequency (%) | – | 0 (45.07) | |||
– | 1 (38.03) | ||||
– | 2 (11.27) |
Scientific name | Source | Accession | Chromosome number |
---|---|---|---|
Zea perennis | CIMMYT | 9475 | 2n = 40 |
Zea mays ssp. mays | USDA | wf9 | 2n = 20 |
Zea mays ssp. mays | USDA | Twf9 | 2n = 40 |
The maize and Z. perennis cytoplasm are represented by light green and light pink circles, respectively. The blue and green strips represent maize chromosomes, while pink, orange, brown and dark red strips represent Z. perennis chromosomes. The centromeres in middle of all chromosomes are labeled red; moreover, both of them have red centromere in the middle. Black parentheses represent paired homologous chromosomes and red parentheses represent expected chromosomes combinations.
In previous studies, two possible evolutionary mechanisms for maize and Z. perennis genome were proposed: First, the genome composition of "AABB” hybrids was an ancestral cross between two closely related "AA” and "BB” genomes that was followed by evolutionary fractionation; Second, Zea species were originated through chromosome duplication, followed by homoeologous genomes "A” and "B” differentiation (
Supplementary tables.